The focus of research in my laboratory is on host-pathogen interactions. Toward that end, we are studying how phagocytes inhibit or kill intracellular microbes using reactive oxygen species (ROS) and nitrogen species (RNS) produced by the NADPH phagocyte oxidase and inducible nitric oxide synthase (iNOS). The specific antimicrobial effector molecules, their targets, and mechanisms of resistance remain incompletely understood. Both the NADPH oxidase and iNOS are required for innate murine resistance to Salmonella infection. Preliminary studies suggest the hypothesis that direct interactions with intracellular free iron determine the antimicrobial actions of ROS and RNS, and regulate relevant stress responses. We propose a novel model in which intracellular free iron potentiates the antimicrobial actions of nitric oxide (NO) and its synergistic interactions with hydrogen peroxide (H2O2). Nitrosative stress induces the expression of iron-repressed proteins such as superoxide dismutase and the Hmp flavohemoprotein via direct NO-iron interactions, which in turn enhance microbial resistance to both ROS and RNS. The specific aims of this proposal are to test predictions of our experimental model by: [1] Comparing Salmonella gene regulation by nitric oxide and iron deprivation; [2] Assessing free intracellular iron as a determinant of susceptibility to ROS and RNS; [3] Performing mutagenesis of the Salmonella Hmp flavohemoprotein to identify domains involved in detoxification of ROS and RNS; [4] Determining the relationship between host and microbial intracellular iron availability and RO S/RNS-dependent antimicrobial activity. These aims will be achieved by a combination of genetic, biochemical and in vivo analyses. The results will have important implications for a molecular understanding of microbial pathogenesis as well as of NO-iron interactions in a variety of fundamental biological processes.